The cell type-specific ER membrane protein UGS148 is not essential in mice

Genomes of higher eukaryotes encode many uncharacterized proteins, and the functions of these proteins cannot be predicted from the primary sequences due to a lack of conserved functional domains. In this study, we focused on a poorly characterized protein UGS148 that is highly expressed in a specialized cell type called tanycytes that line the ventral wall of the third ventricle in the hypothalamus. Immunostaining of UGS148 revealed the fine morphology of tanycytes with highly branched apical ER membranes. Immunoprecipitation revealed that UGS148 associated with mitochondrial ATPase at least in vitro, and ER and mitochondrial signals occasionally overlapped in tanycytes. Mutant mice lacking UGS148 did not exhibit overt phenotypes, suggesting that UGS148 was not essential in mice reared under normal laboratory conditions. We also found that RNA probes that were predicted to uniquely detect UGS148 mRNA cross-reacted with uncharacterized RNAs, highlighting the importance of experimental validation of the specificity of probes during the hybridization-based study of RNA localization

Identification and physiological evaluation of the components from Citrus fruits as potential drugs for anti-corpulence and anticancer

On the basis of monitoring the prevention of accumulation of lipid droplets in mouse 3T3-L1 preadipocyte cells and inhibition of the proliferation of human colon cancer HT-29 cells, effective anti-corpulence and anticancer compounds were isolated from the peel of Citrus fruits. These bioactive components were identified as polymethoxyflavones and coumarin derivatives by spectroscopic analyses. 5-Hydroxy-6,7,8,3′,4′-pentamethoxyflavone had the greatest anti-corpulence effects and 3,5,6,7,8,3′,5′-heptamethoxyflavone had the greatest anticancer effects. Furthermore, distributions of those bioactive components in the peel of 10 species of Citrus fruits were demonstrated by HPLC analyses

Establishment of a Rapid Detection System for ISG20-Dependent SARS-CoV-2 Subreplicon RNA Degradation Induced by Interferon-α

Inhaled nebulized interferon (IFN)-α and IFN-β have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5′-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3′-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 μM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity

The lncRNA Neat1 is required for corpus luteum formation and the establishment of pregnancy in a subpopulation of mice

Neat1 is a non-protein-coding RNA that serves as an architectural component of the nuclear bodies known as paraspeckles. Although cell-based studies indicate that Neat1 is a crucial regulator of gene expression, its physiological relevance remains unclear. Here, we find that Neat1 knockout (KO) mice stochastically fail to become pregnant despite normal ovulation. Unilateral transplantation of wild-type ovaries or the administration of progesterone partially rescued the phenotype, suggesting that corpus luteum dysfunction and concomitant low progesterone were the primary causes of the decreased fertility. In contrast to the faint expression observed in most of the adult tissues, Neat1 was highly expressed in the corpus luteum, and the formation of luteal tissue was severely impaired in nearly half of the Neat1 KO mice. These observations suggest that Neat1 is essential for the formation of the corpus luteum and for the subsequent establishment of pregnancy under a suboptimal condition that has not yet been identified.status: publishe

Time Course Analysis of Skeletal Muscle Pathology of GDE5 Transgenic Mouse

<div><p>Glycerophosphodiesterase 5 (GDE5) selectively hydrolyses glycerophosphocholine to choline and is highly expressed in type II fiber-rich skeletal muscles. We have previously generated that a truncated mutant of GDE5 (GDE5dC471) that lacks phosphodiesterase activity and shown that transgenic mice overexpressing GDE5dC471 in skeletal muscles show less skeletal muscle mass than control mice. However, the molecular mechanism and pathophysiological features underlying decreased skeletal muscle mass in GDE5dC471 mice remain unclear. In this study, we characterized the skeletal muscle disorder throughout development and investigated the primary cause of muscle atrophy. While type I fiber-rich soleus muscle mass was not altered in GDE5dC471 mice, type II fiber-rich muscle mass was reduced in 8-week-old GDE5dC471 mice. Type II fiber-rich muscle mass continued to decrease irreversibly in 1-year-old transgenic mice with an increase in apoptotic cell. Adipose tissue weight and blood triglyceride levels in 8-week-old and 1-year-old transgenic mice were higher than those in control mice. This study also demonstrated compensatory mRNA expression of neuromuscular junction (NMJ) components, including nicotinic acetylcholine receptors (α1, γ, and ε subunits) and acetylcholinesterase in type II fiber-rich quadriceps muscles in GDE5dC471 mice. However, we did not observe morphological changes in NMJs associated with skeletal muscle atrophy in GDE5dC471 mice. We also found that HSP70 protein levels are significantly increased in the skeletal muscles of 2-week-old GDE5dC471 mice and in mouse myoblastic C2C12 cells overexpressing GDE5dC471. These findings suggest that GDE5dC471 mouse is a novel model of early-onset irreversible type II fiber-rich myopathy associated with cellular stress.</p></div

HSP70 protein expression in GDE5dC471 mice.

<p>Total protein from gastrocnemius muscle of 2-week-old and 4-week-old GDE5dC471 mice (Tg) and age-matched control mice (Wild) was subjected to SDS-PAGE followed by Western blotting using anti-GDE5 and anti-HSP70 antibodies. The filter was stained with Coomassie Brilliant Blue (CBB) as a control of protein loading.</p

GDE5dC471 overexpression in skeletal muscle increases neuromuscular junctions-related mRNA expression.

<p><i>A</i>, Total RNA from gastrocnemius muscle of GDE5dC471 mice (Tg) and age-matched control mice (Wild) (4-week-old, n = 4; 8-week-old, n = 3; and 1-year-old, n = 5) was subjected to quantitative PCR to examine mRNA expression level of genes related to neuromuscular junctions. Data represent mean ± SE. *<i>p</i><0.05, **<i>p</i><0.01. <i>B</i>, Representative photographs of fluorescent α-bungarotoxin staining in transverse sections of gastrocnemius muscle, Left; 1-year-old of GDE5dC471 mice (Tg), and right; age-matched control mice (Wild). No morphological alteration of nicotinic acetylcholine receptor (nAchR) was observed between GDE5dC471 and control mice.</p

Obesity-related parameters in 8-week-old and 1-year-old of GDE5dC471 mice, and age-matched control mice.

<p>Obesity-related parameters in 8-week-old and 1-year-old of GDE5dC471 mice, and age-matched control mice.</p